The role of hormone action and hormone replacement therapy in vascular biology is an area of intense interest, but remains highly controversial. Most investigators now favor the hypothesis that the evolving nature of vascular disease makes critical the timing of initiation of estrogen therapy, with a protective effect seen when women receive estrogen during or soon after menopause, rather than much later. It is now well accepted that estrogen has direct effects on cardiovascular tissues that account for the majority of the protective effects of estrogen against vascular diseases. The direct effects of estrogen on the vasculature are mediated by two ligand-activated transcription factors, estrogen receptors ER1 and ER2. This competitive renewal application builds upon over a decade of research done by the Molecular Cardiology Research Institute in collaboration with the Vascular Surgery Division at Tufts-New England Medical Center, in which we have explored the mechanisms of ER1 and ER2 action in the vasculature using wild type (WT) and ER knockout (ERKO) animals, and a combination of molecular, cellular and vascular models. Work to date has refined the understanding of ER action in the vasculature and supports that ERs mediate both control of vascular tone by estrogen and the protective effects of estrogen on vascular injury and atherosclerosis, in part through regulation of NOS isoforms (eNOS, iNOS) by both genomic and non-genomic pathways. However, studies to date have relied on exploring estrogen effects in mouse models in which an ER is disrupted in the early embryo (whole animal ER knockout mice), so that the ER is deleted from conception in all tissues and cells of the body. The present application proposes to test the mechanistic hypotheses we have evolved over the past decade using exciting new genetically modified mice that are now in hand. These mice, and their tissues and cells, enable the precise deletion of ER1 or ER2 from either EC or VSMC and provide powerful approaches to testing the central hypotheses of this application that vascular endothelial cell ER1 regulates eNOS expression and activity; vascular smooth muscle cell (VSMC) ER1 mediates estrogen inhibition of VSMC proliferation during vascular injury and atherosclerosis; and VSMC ER2 controls genes encoding key contractile proteins regulating VSMC tone. These mechanisms are explored in 3 Specific Aims using intact animals, blood vessels and cells with VSMC- or EC-specific, induced deletions of ER1 or ER2 in studies of the (SA1) role of vascular ER1 and ER2 in the regulation of vascular tone in intact vessels; (SA2) role of vascular ER1 and ER2 in estrogen protection of the vascular injury response in normolipidemic animals; and (SA3) role of vascular ER1 and ER2 in atherosclerosis. These studies are expected to add substantially to our understanding of the molecular and cellular biology of ERs expressed in the blood vessel and vascular physiology, with important impact on the field of women's health and on the development of new therapies for the prevention and treatment of vascular diseases in both women and men.Project Narrative This proposal will explore in depth how the hormone estrogen directly influences blood vessels in health and disease. The studies use state-of-the-art methods to study questions about how estrogen is protective against heart disease, and why recent studies of hormone replacement therapy in women raised concerns about estrogen in women well past the menopause. These studies are expected to have an important and substantial impact on the diagnosis of vascular diseases, on the design of new therapies for the prevention and treatment of vascular diseases, on women's health, and on public health in general.